We are using human erythroid K562 cells as a model to try to understand the function of enzymatic activities that modify histones. Although H3K9me2 and me3 are typically features of silent heterochromatin, we and others found H3 K9 tri-methylation associated with actively transcribed globin genes. Further, G9A, the methyl-transferase responsible for euchromatic lysine 9 dimethylation, is recruited to the beta-globin LCR and active globin gene promoters. To determine whether G9A and/or its partner methyltransferase GLP are responsible for the H3K9me3 in actively transcribed globin genes, we reduced expression of these proteins singly and concurrently in erythroid cells using RNAi. We are examining the consequences of these protein knock downs for globin transcription and localization of HMTase complex components. In addition, we will knockdown the K4 HMT complex component Ash2L and examine the effect on transcription and K4 and K9 methylation to determine whether K4 and K9 methylation marks are functionally related. How individual globin genes establish stage specific enhancer communication is unknown and most studies have been performed in a non-chromosomal environment. We are using homologous recombination in mouse ES cells to address this question. We targeted the mouse embryonic epsilon y gene in ES cells and then used recombinase mediated cassette exchange to replace it with epsilon y genes in which selected promoter transcription factor motifs had been destroyed or altered. Most mutations modestly perturbed epsilon y transcription, indicating that, individually, they are dispensable for LCR/epsilon communication. Substitution of the beta-globin CACCC site was a strong up-regulator of epsilon y transcription and resulted in recruitment of the adult specific factor EKLF to the promoter, consistent with an important role for the CACCC element in stage specific globin gene expression. These experiments are novel since they alter transcription factor recruitment in a normal chromosomal setting and will provide information on how the LCR and globin genes communicate in vivo.